Our overall purpose is to take aim at two urgent clinical problems for persons with spinal cord injury (SCI). The first problem is ongoing morbidity from urinary tract infections (UTI) and mortality from bacteremia and sepsis arising from pathogens in the urinary tract. Urinary tract infections and associated complications are a common threat to the wellbeing and even survival of persons with spinal cord injury (SCI). The second clinical dilemma is the disproportionate harm persons with SCI are experiencing from multidrug -resistant bacteria, particularly Escherichia coli, the most common urinary pathogen. We plan to develop bacteriophage, viruses that kill specific strains of bacteria, as a treatment for multidrug-resistant E. coli UTI and sepsis in SCI. Phage have numerous properties that suggest they will be effective in this role, including lack of cross resistance with antibiotics, ability to attack biofilms, evolvability in vitro, and being a self-renewing agent at the site of infection. Phage therapy for resistant urinary organisms has potential applications in the SCI population beyond UTI treatment and also for the Veteran population in general, such as for bladder sterilization prior to urologic procedures. Phage therapy aligns with VHA antibiotic stewardship efforts and may alleviate the burden of Clostridium difficile experienced by Veterans. The work we propose here with the most common urinary pathogen in SCI, E. coli, would subsequently be applied to the other SCI urinary pathogens to create broadly effective phage cocktails. Project Objectives: We will develop E. coli-specific phages that are efficacious against a broad range of SCI-specific E. coli isolates from our VA hospital in a mouse model of catheter-associated urinary tract infection (CAUTI) and/or sepsis. Aim 1 is to determine the efficacy of phage in killing antibiotic-resistant E. coli in murine infection models of CAUTI (1A), bacteremia (1B), and in combination with antibiotics to which the infecting E. coli pathogen is resistant (1C). Aim 2 is to develop and test a bank of phages effective against contemporary E. coli isolates from Veterans with SCI. In Aim 2A, we will actively collect E. coli clinical isolates from Veterans with SCI, creating an SCI-specific bacterial strain bank. In Aim 2B, we will collect and experimentally generate phages with broad host range against E. coli, creating a bank of sequenced phage that cover over 80% of the SCI E. coli strains. In Aim 2C we will create phage cocktails from our phage bank, and test the efficacy of the cocktails in the mouse model of CAUTI induced by SCI E. coli. Aim 3 is to examine the emergence of phage-resistant E. coli during treatment of CAUTI and determine how to overcome phage resistance. Aim 3A: Determine if E. coli persistence after phage treatment results from bacterial resistance to phage. Aim 3Bb: Determine if a ?recharge? dose of phage several days into therapy prevents recurrence of E. coli. Methods: We propose to study bacteriophage in vitro and in vivo using our established models of mouse CAUTI and bacteremia for all three aims. In these experiments we will track microbiological outcomes and clinical outcomes. The microbiological outcomes include quantitative counts of bacteria and phage, [microbiome diversity before and after treatment with phage and/or antibiotics, and images of urinary catheter-associated biofilms.] Our clinical outcomes are intended to recapitulate the human experience, particularly the ability to perform activities of daily living. We have two measures of clinical outcomes: the disease severity index (assesses health) and the behavioral score (assesses whether animals still perform routine activities). The experiments in Aim 1 will use bacteria (E. coli JJ2528) and phage (?HP3) from our existing collections, so that the work can begin immediately. At the end of this proposal, we will have a bacterial strain bank specific to SCI, and we also will have developed, characterized, and tested in vivo a library of phage that are effective at lysing over 80% of these SCI isolates. Our team brings together complementary expertise in clinical infections, epidemiology, SCI clinical management, SCI research, microbiology, virology, [metagenomics, biofilm imaging,] and animal studies to accomplish the work proposed.